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MSX1 Gene is Deleted in Wolf-Hirschhorn Syndrome Patients with Oligodontia
1 Institute of Dentistry, Biomedicum, PO Box 63, FIN-00014 University of Helsinki, Finland; Correspondence: *corresponding author, pekka.nieminen{at}helsinki.fi
Abnormalities of the short arm of chromosome 4 cause multiple congenital malformations, including craniofacial, oral, and dental manifestations. A candidate gene for oral defects in this region is MSX1, which is mandatory for normal oral and tooth development. We examined the dentition and the presence of MSX1 in eight Finnish patients with abnormalities of 4p, including seven cases of Wolf-Hirschhorn syndrome. Five of the Wolf-Hirschhorn syndrome patients presented with agenesis of several teeth, suggesting that oligodontia may be a common (even though previously not well-documented) feature in Wolf-Hirschhorn syndrome. In fluorescence in situ hybridization (FISH) analysis, the five patients with oligodontia lacked one copy of MSX1, while the other three had two hybridization signals. One of these presented with the only case of cleft palate among the patients. Our result confirms that haploinsufficiency for MSX1 serves as a mechanism that causes selective tooth agenesis but, alone, is not enough to cause oral clefts.
Key Words: MSX1 • oligodontia • tooth agenesis • cleft palate • Wolf-Hirschhorn syndrome
Wolf-Hirschhorn syndrome (OMIM 194190, http://www3.ncbi.nlm.nih.gov/omim/) is a multi-organ syndrome caused by deletions of the short arm of chromosome 4 (4p), classifying Wolf-Hirschhorn syndrome as a contiguous gene syndrome (Lurie et al., 1980; Morishita et al., 1983). The deletions may be a result of a translocation or ring chromosome formation. The manifestations include mental and growth retardation, craniofacial abnormalities, seizures, and hypospadia. Overlapping deletions have led to a suggestion of a relatively short critical region of fewer than 200 kb (Wright et al., 1997). However, relationships of phenotypic variability and the extent of deleted chromosomal material have remained poorly defined and controversial (Estabrooks et al., 1995; Wieczorek et al., 2000; Zollino et al., 2000). The craniofacial abnormalities include microcephaly, maxillary hypoplasia, hypertelorism, high nasal bridge with a characteristic Greek warrior helmet appearance, oral clefts, and dental anomalies (Morishita et al., 1983). The dental abnormalities reported include delayed development and fusion of incisors (Burgersdijk and Tan, 1978; Morishita et al., 1983; Kotilainen, 1996). A single case with congenital tooth agenesis has been described (Burgersdijk and Tan, 1978), but, as noted by the authors, it is conceivable that tooth agenesis may be much more common among the syndrome patients because of problems in the diagnosis of dental anomalies, especially with young patients. An obvious candidate gene for oral and dental defects is MSX1, which is located about 3 MB proximal to the critical region for Wolf-Hirschhorn syndrome and may be involved in the larger deletions or re-arrangements (Ivens et al., 1990; Entrez Human Genome, NCBI, http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/hum_srch). MSX1 codes for a transcription factor that is expressed in dental mesenchyme and regulates signaling and tissue interactions during early stages of tooth development (Vainio et al., 1993; Chen et al., 1996). Msx1 has been shown to be mandatory for normal oral and tooth development in the mouse (Satokata and Maas, 1994). In man, mutations in MSX1 have been reported in families with dominantly inherited congenital absence of several permanent teeth (oligodontia) (Vastardis et al., 1996; van den Boogaard et al., 2000; Jumlongras et al., 2001; Lidral and Reising, 2002). In one family, some of the patients also had various types of oral clefts (van den Boogaard et al., 2000), while in another family a nail dysplasia was described (Jumlongras et al., 2001), suggesting a general role for MSX1 in the development of ectodermal derivatives. Polymorphisms in MSX1 have also been associated with oral clefting (Lidral et al., 1998; Jezewski et al., 2003), and in a large group of patients with oral clefts, several sequence alterations in MSX1 were recently reported (Jezewski et al., 2003). However, congenital absence of a few incisors or premolars in five Finnish families was not linked to a polymorphism within MSX1 (Nieminen et al., 1995). We studied eight Finnish patients with deletions in the short arm of chromosome 4 (4p). Five of these patients, two of whom were twins, had severely affected dental development, resulting in agenesis of several teeth. As revealed by fluorescence in situ hybridization (FISH), lack of one copy of MSX1 was completely associated with oligodontia among these patients, thus confirming the conclusion that selective tooth agenesis caused by MSX1 loss-of-function mutations is caused by haploinsufficiency.
Subjects We studied eight individuals with a deletion in the short arm of chromosome 4 (Table 1  ). Seven of them had been diagnosed as Wolf-Hirschhorn syndrome patients (patients 2 through 8 in Table 1) while one (patient 1) was included in the study because of the presence of a 4p deletion. Some of the patients (#7 and 8) have been described earlier (Kotilainen, 1996). Five of the patients were participating in a rehabilitation course for families with Wolf-Hirschhorn (del 4p) syndrome. Three of the patients were receiving services at the Rinnekoti Central Institute. Four of the patients were females, four males, and their ages ranged from 5.8 to 21.7 yrs. Initial diagnoses had been made in the University Hospitals of Helsinki, Turku, or Tampere and been confirmed in early childhood with cytogenetics or fluorescence in situ hybridization (FISH). Ages and karyotypes of the patients are included in Table 1. The study was conducted with the consent of the parents and the Rinnekoti Central Institute and approved by the Ethics Committee of the Institute of Dentistry, University of Helsinki.
Dental and Oral Examinations Congenitally missing teeth were assessed from panoramic radiographs. Of two patients from Rinnekoti Central Institute, two panoramic radiographs with several years’ interval were available (Table 1  ). In case of obscure regions in radiographs, the number of erupted teeth was confirmed in clinical examination. Signs of enamel hypoplasia and severely worn dentition were also recorded. The anatomy of the palate and structure of hair and nails were recorded in a clinical examination or obtained from patient records.
FISH Analysis
Tooth agenesis of several permanent teeth were observed in five of the patients (patients 3, 4, 5, 6, and 7; see Table 1 , Fig. a). Additionally, one of the other patients (#8) lacked three third molars. Common to the five patients, the posterior teeth were more severely affected, while some anterior teeth were also always missing. Most constantly affected were the second premolars, which were completely missing in all patients. Third molars could not be seen in any of the five patients. However, in several cases (#1, 2, 3, 4, and 6), definitive diagnosis of congenitally missing third molars was not possible because of the young age of the patients. In only one case (patient 5) could we detect some missing primary teeth, in this case two lower lateral incisors. All five patients with multiple congenitally missing teeth also had either severely worn dentition (#5, 6, and 7; Figs. b,c) or enamel hypoplasia with brownish hypoplastic permanent incisors (patients #3, 4, and 5).
One patient, #8, had cleft palate (Table 1 ). Three patients (#1, 6, and 7) had a high narrow palate. Two of them also had oligodontia. All other patients had a normal palate. No nail or hair abnormalities were observed or registered in the patient records.
FISH analysis was performed with two different MSX1 genomic clones which gave consistent results (Table 1
In this report, we have described the congenital absence of several teeth in five Finnish Wolf-Hirschhorn syndrome patients. To our knowledge, congenital absence of teeth in patients with this syndrome has been described in only one report (Burgersdijk and Tan, 1978). As suggested by these authors, it is possible that tooth agenesis may have been overlooked by non-dental professionals. In most cases, the patients have been so young that agenesis of permanent teeth would have been possible to detect only from x-rays. Also, because of the characteristics of the patients, both clinical and radiological examination is often difficult. Our finding suggests that oligodontia may be considered a common feature in Wolf-Hirschhorn syndrome and could be used as an indication. A panoramic radiograph of dentition should be taken of Wolf-Hirschhorn patients over five years of age to diagnose congenitally missing permanent teeth. In the case of missing permanent teeth, corresponding primary teeth should serve for the entire lifetime.
We have also shown an absence of one copy of the MSX1 gene in five of the patients. The critical region for Wolf-Hirschhorn syndrome has been delineated to a location of 1.9 Mb from the telomere of the short arm of chromosome 4, and MSX1 is located about 3 Mb proximal to that. Our results are in good agreement with earlier chromosomal analyses and help to fine-map the extent of the deletions (Table 1
The importance of the MSX1 deletion in the etiology of oligodontia in the five patients is underlined by a high resemblance of the phenotypes to oligodontia in families with MSX1 mutations, published earlier (Vastardis et al., 1996; van den Boogaard et al., 2000; Jumlongras et al., 2001; Lidral and Reising, 2002). In these four families and in the patients described here (Table 2
The MSX1 mutations described earlier have all been considered as loss-of-function mutations that, in the heterozygous state, lead to a decreased amount of functional protein, i.e., haploinsufficiency (Vastardis et al., 1996; Hu et al., 1998; van den Boogaard et al., 2000; Jumlongras et al., 2001; Lidral and Reising, 2002). The deletion of MSX1 in Wolf-Hirschhorn syndrome patients presumably leads to complete MSX1 haploinsufficiency. In accordance, the oligodontia phenotypes of the Wolf-Hirschhorn syndrome patients are generally more severe than in most of the families described earlier. They most closely resemble the phenotypes associated with a Ser202Stop mutation (Jumlongras et al., 2001). In both patient groups, the absence of, especially, lower first permanent molars and upper first premolars was nearly complete (Table 2 ). The mild hypodontia of one of the patients (#8) with both copies of MSX1 is most probably explained by factors other than decreased expression of MSX1. Congenital agenesis of at least one of the third molars is rather common, with a prevalence of over 20% (Grahnen, 1956; Haavikko, 1971), and it may have multiple genetic or environmental reasons. However, it cannot be excluded that even though the patient had both copies of MSX1, the regulation of gene expression is altered because of the nearby deletion or the consequences of the ring chromosome formation. In the mouse, Msx1 is required for normal development of the palate (Satokata and Maas, 1994), and in man it has also been implicated in palatal development (Lidral et al., 1998; van den Boogaard et al., 2000; Jezewski et al., 2003). In our patient group, the only case of cleft palate was found in a boy with both copies of MSX1, while a milder abnormality of high and narrow palate was observed in three patients, two of whom had MSX1 haploinsufficiency. This shows directly that MSX1 haploinsufficiency alone does not lead to abnormal palatal development. Further, we did not detect any abnormalities of nails or hair, suggesting that normal amounts of the MSX1 protein are not critical for development of these ectodermal derivatives. Our finding that the patients with MSX1 haploinsufficiency also had enamel hypoplasia or severely worn dentition raises the question whether MSX1 also affects the differentiation and mineralization of dental hard tissues. It is conceivable that worn dentition was secondary to the oligodontia. Since MSX1 is not known to be expressed in ameloblasts or their predecessors, and since enamel defects are a rather common finding, the enamel defect in the two patients probably has different etiology. In addition to MSX1, mutations in PAX9 can lead to isolated tooth agenesis (Stockton et al., 2000; Nieminen et al., 2001; Das et al., 2002, 2003; Frazier-Bowers et al., 2002; Lammi et al., 2003). In the families where loss-of-function mutations of PAX9 have been identified, oligodontia characteristically affects permanent molars in addition to a variable number of other teeth. In most patients with PAX9 mutations, a missing second premolar or a first permanent molar occurs only when all the posterior molar teeth are also missing. However, in some patients with MSX1 or PAX9 mutations, the phenotypes are rather similar. It is possible that different types of mutations in each gene result in different phenotypes. The similarities of phenotypes may also be explained by the observation that, in the mouse, Pax9 apparently also regulates Msx1 expression (Peters et al., 1998). However, since the mouse Msx1 or Pax9 null mutant heterozygotes have normal dentition (Satokata and Maas, 1994; Peters et al., 1998), it is obvious that tooth development has a dose-sensitivity for both genes which is demonstrated during the development of human, but not mouse, dentition. In summary, our results suggest that selective tooth agenesis is a common phenotype in Wolf-Hirschhorn syndrome. Oligodontia is associated with deletion of one copy of the MSX1 gene, supporting the conclusion that tooth agenesis associated with mutations in MSX1 is caused by haploinsufficiency. Our finding represents the most striking correlation of a specific phenotype with the extent of deletion outside the critical region for Wolf-Hirschhorn syndrome reported so far.
We are grateful to Dr. Eero Palolampi for his permission to examine patients, and to the staff of the Rehabilitation Center for Sick Children of the Mannerheim League, Finland, for practical arrangements. We thank Dr. Markus Kaski and Dr. Carola Tengström from the Rinnekoti Central Institute for their collaboration. The MSX1 probe was a gift from Dr. Jane E. Hewitt, University of Manchester. The skillful help of the late Ms. Kaija Kettunen is thankfully acknowledged. We are also grateful to Dr. Sirpa Arte and Dr. Mirja Somer for their critical comments on the manuscript. This work was supported by the Academy of Finland and the Finnish Cultural Foundation. Received for publication March 19, 2003. Revision received August 27, 2003. Accepted for publication September 17, 2003.
Journal of Dental Research, Vol. 82, No. 12,
1013-1017 (2003) This article has been cited by other articles:
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